Lonq-Term Objectives: Elucidate fundamental relationships of chemical catalysis, enzyme conformational dynamics, allosteric regulation, and enzyme structure to the mechanism of metabolite transfer between enzyme pairs (substrate channeling) in a metabolic cycle. Specific Aims: To achieve a detailed mechanistic description of the allosteric regulation of substrate channeling in the bacterial tryptophan synthase bienzyme complex. The roles played by the binding of allosteric effectors, the formation of intermediates, and the binding of a monovalent cation cofactor in the regulation of channeling will be determined. Backqround: The tryptophan synthase bienzyme complex catalyzes the last two steps in the biosynthesis of L-tryptophan. Catalysis is intimately related to allosteric signaling and metabolite transfer between the alpha- and beta-sites. Hypotheses To Be Tested: (a) The alpha- and beta-subunits of the bienzyme complex switch between "open", "partially open" and "closed" conformation states during the catalytic cycle in response to binding and covalent reaction of substrates. (b) Certain covalent states of the beta-site give rise to a slow switching from the closed to the open conformation state(s) that is functionally important for substrate channeling. (c) Monovalent cation (MVC) binding plays a cofactor role that modulates the catalytic and conformational energetics of the complex. (d) The BetaAsp305-BeataArg141 salt bridge is critically important for the allosteric regulation and reaction specificity of the complex. Methods: The hypotheses will be tested via the use of rapid kinetics, equilibrium binding, 19F NMR, mutant enzymes, substrate analogues, optical spectroscopy and x-ray crystallography. The project Significance to Human Health and Disease derives from the new knowledge about structure-function relationships for the class of protein nanostructures consisting of enzyme-enzyme complexes that are organized into metabolic pathways. These nanostructures are subject to non-traditional forms of allosteric regulation that are poorly understood. Four project areas are of fundamental interest: (a) The roles of protein structural elements and protein conformational dynamics in substrate channeling between enzyme pairs in metabolic pathways. (b) The roles played by protein conformational dynamics in biological function, (c) the roles of monovalent cations in the modulation of protein function, (d) the role played by substrates as allosteric effectors in substrate channeling.